Penetrating investigations into the pathogenesis of neurological SIDS have implicated a developmental defect in the medullary serotonergic (5-HT) system, defined as an interrelated group of 5-HT and non-5-HT neurons centered around the medullary raphe. Unfortunately, littte is known about the molecules during differentiation of these neurons and how their development is affected by environmental SIDS risk factors -information that is critical for determining the etiology of SIDS. To help fill this void, studies proposed in Project 6 focus on testing in mice the hypotheses that neurons of the medullary 5-HT system come from the rhombic lip ; that their identities are specified, at least in part, as precursor cells in the rhombic tip, and that abnormalities in these early specification events underlie the medullary dysfunction associated with SIDS. In aim 1, we use a unique set of generic tools to establish that neurons of the medullary 5-HT system come from the rhombic lip; indeed, our preliminary fate maps indicate this is the case. We extend these analyses to characterize the dispersion of 5-HT system neurons from the rhombic lip and map their acquisition of different neurochemical identities. In most neuroepithetia, including the rhombic lip, the potential to give rise to different neuron types appears to change over time, with restrictive events occurring in the dividing progenitor cells themselves. Thus, at distinct developmental stages a given neuroepithelium is multipotent but not equipotent, presumably due in part to temporal changes in intrinsic regulators. To identify such regulators in the rhombic lip, in aim 2, we perform whole-genome expression studies of rhombic lip tissue at sequential developmental stages corresponding to its peak production times of different brainstem neurons. By comparing mRNA profiles across time points, we will identify gene sets associated with neuron subtype production. To further sift from these gene sets the best candidate regulators for future functional studies, we perform similar analyses on tissue from mice null for Pax6, a transcription factor required for producing a subset of rhombic-lip derivatives and an established regulator of CNS fate. From this, we identify mRNAs dependent on Pax6 and which may be important for a subset of 5-HT-system specification events. In aim3, we determine if prenatal exposure to nicotine (a major component of cigarette smoke and proven neuroteratogen) alters development of the 5-HT system in ways that may explain the increased risk for SIDS following maternal smoking. Completion of these aims will provide a comprehensive molecular basis for delineating brainstem development and for identifying mechanisms underlying SIDS.